1. Field of the Invention
The present invention relates to a method for obtaining eukaryotic organisms, i.e. plants, animals or fungi, with elevated activity and/or altered regulatory capacity of trehalose-6-phosphate synthase. The invention also relates to specifically modified alleles of the trehalose-6-phosphate synthase genes which display unexpected changes in catalytic activity and/or regulatory capacity, and to the transformed plants, or other eukaryotic organisms containing these constructs. The invention is also related to novel methods to measure the level of trehalose-6-phosphate and the activity of trehalose-6-phosphate synthase.
2. Description of the Related Art
The biosynthesis of trehalose consists of two enzymatic steps catalyzed by trehalose-6-phosphate synthase (TPS), which synthesizes trehalose-6-phosphate, and by trehalose-6-phosphate phosphatase (TPP), which forms trehalose. The genes of the trehalose metabolism have been discovered first in yeast and in bacteria, organisms that were known for a long time to accumulate trehalose. Recently, homologues of these genes have also been found in higher plants and animals in which appreciable levels of trehalose had never been detected. However, up to now it has not been possible to demonstrate enzymatic trehalose-6-phosphate synthase activity of these TPS gene products in any in vitro system. Their expression in heterologous systems also does not result in high trehalose accumulation. No successful usage of these plant or animal TPS genes to improve commercially important properties in homologous or heterologous systems has been reported.
In addition to its classical role in storage sugar accumulation, trehalose metabolism is known to play important roles in stress resistance, control of glucose influx into glycolysis and glucose-induced signalling. As outlined below, these phenotypic properties are of high industrial importance.
An amazing capacity for adaptation to survival under strong or even complete dehydration is present in yeast cells, fungal spores, certain invertebrate species and resurrection plants, which resume their vital functions as soon as they are again in contact with water. These anhydrobiotic organisms also withstand freezing, strong vacuum, high doses of ionizing radiation, high pressure and extreme temperatures without suffering damage and many of them accumulate the non-reducing disaccharide trehalose as a protein and membrane protectant.
The protectant function of trehalose has also been demonstrated in vitro. Addition of trehalose to cells, organelles, enzymes, antibodies and foods preserves them under total dehydration for long periods. It also protects them against a variety of other stress conditions, such as high temperature, high pressure and freezing.
In vascular plants, very few species are known where the presence of trehalose has been demonstrated in a convincing way. However, in the so-called desert resurrection plant Selaginella lepidophylla a high trehalose level is present. This plant is able to withstand successfully complete dehydration, as opposed to all other higher plants including crop plants.
Deletion mutants in the TPS gene in bacteria and yeast are unable to synthesize trehalose and they lose osmotolerance, thermotolerance and tolerance to high pressure. This suggests that the TPS gene is involved in various forms of tolerance.
It would be highly desirable to be able to express trehalose-6-phosphate synthase activity in plants, animals, micro-organisms or specific parts thereof in order to render them tolerant to stress. In this way, crop plants could be cultured in regions suffering occasionally or continuously from heat, drought or freezing. Perishable foods from plant or animal origin could be preserved by simple dehydration, enabling storage over a prolonged period of time and transport long distances.